Summary The loss of regenerative capacity during aging is a major unsolved problem in biology and biomedicine. Here we continue to develop the tunicate Ciona intestinalis as a model for regenerative aging research. Ciona has many favorable attributes for this purpose, including a small sequenced genome, an extensive tool kit for genetic manipulations, a short life span, powerful regeneration capacities that disappear with age, an identified stem cell niche involved in regeneration, a collection of stable transgenic lines with green fluorescent protein expressed in specific tissues, and the ability to produce chimeric animals containing young and old body parts. Most importantly, Ciona belongs to an invertebrate chordate group recognized as the closest living relative of vertebrates, suggesting that research on this organism will be generally relevant to vertebrates, including humans. The three specific aims of this proposal are designed to incrementally advance our understanding the relative roles of the stem cell niche, the blastema, and the surrounding tissue environment in regenerative aging and the molecular mechanisms governing this process using the Ciona oral siphon (mouth) as a model. The first aim will use a novel chimera assay in which stem cell niches are swapped between young and old animals and isolated stem cells are injected into old animals to determine the relative roles of the stem cell niche, the stem cells themselves, the regeneration blastema, and the surrounding tissue environment in regenerative aging. This aim will exploit our previous identification of the branchial sac (pharynx) stem cell niche as the source of progenitor cells that migrate the site of siphon amputation and differentiate into multiple tissues during regeneration. The second aim is to identify, characterize, and localize the expression of key regulatory genes that are responsible for robust siphon regeneration in young animals and loss of this capacity in old animals. This aim will use both a set of pre-existing candidate genes expressed at high levels in the regenerating siphon of young animals as well as a new set of downregulated and upregulated regulatory genes expressed in the stem cell niche to be identified by transcriptome sequencing at different stages of the adult life cycle. The third aim is to determine the functions, hierarchical relationships, and ability to restore lost regenerative capacity of key regeneration genes by RNAi mediated gene knockdown and amelioration assays using the chimera approach in old animals. This research is designed to reveal the genetic mechanisms underlying the loss of regenerative capacity during aging in an invertebrate chordate model with the potential to clarify our understanding of the evolutionary history and causes of regenerative aging in vertebrates.